The first unique step leading to DNA synthesis is the conversion of ribonucleotides to their corresponding deoxyribonucleotides, a reaction that is catalyzed in a cell cycle specific manner by the housekeeping gene ribonucleotide reductase [Lewis et al., 1978; Reichard, 1993; Wright, 1989a; Wright et al., 1990a; Stubbe, 1989]. The mammalian enzyme is composed of two dissimilar dimeric protein components often called R1 and R2, which are encoded by two different genes located on different chromosomes [Björklund et al., 1993; Tonin et al., 1987]. Mammalian protein R1 is a homodimeric structure, with a molecular weight of about 170 kDa, and has substrate sites and allosteric effector sites that control enzyme activity and substrate specificity [Wright, 1989; Thelander et al., 1980; Caras et al., 1985; Wright et al., 1990a]. Protein R2 is a homodimer, with a molecular weight of 88 KDa, and forms two equivalent dinuclear iron centers that stabilizes a tyrosyl free radical required for catalysis [Wright et al., 1990a; Thelander et al., 1985; McClarty et al., 1990]. R1 and R2 proteins interact at their C-terminal ends to form an active holoenzyme [Reichard, 1993; Wright et al., 1990a; Davis et al., 1994].
R1 and R2 are differentially regulated during the cell cycle. There is an S-phase correlated increase in the R2 protein resulting from its de novo synthesis [Lewis et al., 1978; Mann et al., 1988]. The activity of ribonucleotide reductase, and therefore DNA synthesis and cell proliferation, is controlled in proliferating cells during the cell cycle by the synthesis and degradation of the R2 component [Eriksson et al., 1984]. The rate-limiting R2 component is a phosphoprotein capable of being phosphorylated by the CDC2 and CDK2 protein kinase mediators of cell cycle progression [Chan et al., 1993], and contains non-heme iron that stabilizes an unique tyrosyl free radical required for enzyme activity [Reichard, 1993; McClarty et al., 1990].
The levels of the R1 protein do not appear to change substantially during the cell cycle of proliferating cells and can be detected throughout the cell cycle. Synthesis of R1 mRNA, like R2 mRNA appears to occur mainly during S phase [Eriksson et al., 1984; Choy et al., 1988; Mann et al., 1988]. The broader distribution of the R1 protein during the cell cycle is attributed to its longer half life as compared to the R2 protein [Choy et al., 1988; Mann et al., 1988.
Regulation of ribonucleotide reductase, and particularly the R2 component, is markedly altered in malignant cells exposed to tumor promoters or to the growth factor TGF-β [Amara, et al., 1994; Chen et al., 1993; Amara et al., 1995b; Hurta and Wright, 1995; Hurta et al., 1991]. Higher levels of enzyme activity have been observed in cultured malignant cells when compared to nonmalignant cells [Weber, 1983; Takeda and Weber, 1981; Wright et al., 1989a], and increased levels of R2 protein and R2 mRNA have been found in pre-malignant and malignant tissues as compared to normal control tissue samples [Saeki et al., 1995; Jensen et al., 1994]. Regulation of ribonucleotide reductase, and in particular the R2 component, is significantly elevated in transformed cells exposed to tumor promoters, or to transforming growth factor β in growth factor mediated mechanisms of tumor progression [Amara et al., 1996; Chen et al., 1993; Amara et al, 1995b]. These studies are in tumor cells obtained from rodent and human tissues [Weber, 1983; Wright et al., 1989a; Saeki, et al., 1995; Jenson et al, 1994], and in cultured cells selected for resistance to anti-tumor agents such as hydroxyurea [Lewis et al., 1978; Wright et al., 1989b].
Chemotherapeutic compounds like hydroxyurea inhibit ribonucleotide reductase activity by destabilizing the iron centre of the R2 protein causing the destruction of the tyrosyl free radical [McClarty et al., 1990], and preventing cells from progressing through S-phase of the cell cycle [Ashihara and Baserga, 1979]. In addition to cell cycle control, ribonucleotide reductase can be regulated by an S-phase independent mechanism that is important for DNA repair. Ribonucleotide reductase activity can be induced outside the S phase by DNA cross-linking agents such as chlorambucil, and by UV irradiation indicating a role for the enzyme in the DNA repair process [Hurta and Wright, 1992; Filatov, et al., 1996].
Recent studies have shown that ribonucleotide reductase activity is quickly elevated in the presence of tumor promoters like 12-0-tetradecanoylphorbol-13-acetate [Amara et al., 1994; Chen et al., 1993]. This process is mediated at least in part, through increases in the half-lives of R1 and R2 mRNAs, which parallels the decreased interactions of two proteins, R1BP and R2BP, with cis-element sequences in the 3′ untranslated regions (3′ UTRs) of the R1 and R2 messages [Amara et al., 1994; Chen et al., 1993; Chen et al., 1994a; Chen et al., 1994b]. Alterations in this cis-trans reaction can play a role in determining sensitivity to chemotherapeutic agents that target ribonucleotide reductase [Amara et al., 1995a].
Exposure of transformed fibroblasts to TGF-β1 can increase the half-life of the R2 message, a process that is mediated through a cis-trans interaction within the R2 mRNA 3′ UTR [Amara et al., 1995b; Hurta and Wright, 1995]. Other studies have demonstrated that the non-coding regions of mRNAs can control important biological properties of cells, such as the expression of bFGF in Xenopus oocytes [Kimelman and Kirschner, 1989], the timing of developmental events of Caenorhabditis elegans [Lee et al., 1993], the expression of α1 (I) collagen in chick embryo chondrocytes [Farrell and Lukens, 1995], and the suppression of tumorigenicity of rhabdomyosarcomas by RNA from the 3′ UTR of the non-housekeeping gene α-tropomyosin [Rastinejad et al., 1993].
PCT patent application WO 94/21661 discusses the use of UTRs of cell structural proteins to regulate cell division or cell differentiation and provides a discussion of how exogenous UTR may affect cell regulation. The application in particular relates to UTRs of α-tropomyosin.
The regulation of mRNA turnover is an essential step in controlling message abundance and therefore gene expression in mammalian cells. Message degradation or stability plays a critical role in cell proliferation or cellular differentiation, and is crucial in mechanisms that maintain normal biological functions of individual cells and tissues. Aberrant mRNA turnover usually leads to altered levels of proteins, which can dramatically modify cellular properties. For example, oncogene or growth factor overexpression is often associated with abnormal cell proliferation and malignant transformation. Since message turnover is an important component of gene regulation, it is not surprising to find that message stability characteristics of key growth regulatory genes are tightly controlled. Several excellent reviews are available which describe in detail mechanisms of gene expression that are regulated at the mRNA level [Ross, 1995; Hake and Richer, 1997].
Messenger RNA is composed of distinct domains that either encode proteins or carry specific regulatory regions that control gene expression posttranscriptionally. Structurally there are three distinct regions of an mRNA molecule, the 5′ end including the cap (5′-GpppG--), the coding region, and the 3′ end including the polyadenylated tail. The structural elements of mRNA are known to play integral roles in mechanisms regulating translation and mRNA stability, which in turn directly affect translation efficiency and the turnover rate of the message, and therefore the amount of a specific protein that is synthesized.
The 5′ end of an mRNA molecule contains a sequence that is not translated into protein and therefore is known as the 5′ untranslated region (UTR), and contains the mRNA cap which confers nuclease resistance properties. There is a great deal of evidence showing that the 5′ end of a message is critically involved in regulating translation initiation [Ross, 1995; Hake and Richer, 1997]. Alterations in translation regulation not only directly affects the amount of a protein that is eventually synthesized, but it can also significantly modify the stability characteristics of the message and therefore modify protein levels by this mechanism as well. For example, some viruses are capable of modifying the binding of regulatory proteins to the 5′ UTR including the cap region, and through this process control host versus virus gene expression. The 5′ UTR of a message can be relatively short or can be several hundred nucleotides in length.
There is also a region of varying length following the coding sequence that is not translated into protein, and this 3′ UTR which may be many hundreds of nucleotides in length, appears to play a dominant role in determining message stability characteristics. There are now many examples of unique cis-elements in this part of the message that bind to trans-acting proteins to control mRNA turnover rates [Ross, 1995; Hake and Richer, 1997].
In addition, most mRNAs have a polyadenylated (poly (A)) tail at the 3 end, which can serve several functions important to translation efficiency and message turnover characteristics. For example the poly (A) tail protects the message from degradation in some systems, and it has been demonstrated that deadenylation may be the first step in message degradation. The mere presence of a poly (A) tail is not necessarily sufficient for protection, instead the poly (A) tail should be a minimum length, for example 20 to 30 nucleotides long, to provide protection from nuclease action. When the number of residues is changed experimentally, the rate of degradation can be increased or decreased by the absence or presence of a specific number of residues. Several proteins are involved in this regulation including a poly (A) binding protein, and it has been suggested that the poly (A) tail blocks the assembly of an exonuclease involved in RNA degradation [Sachs, 1993; Ford et al., 1997].
Besides the interactions between cis-elements with precise nucleotide sequences and trans-acting proteins, secondary structural conformations such as stem-loops and hairpin structures also serve regulatory functions in the untranslated regions (UTRs) of mRNAs. For example, it has been shown in some cases, that it is possible to transfer sequences containing interesting structural features from the UTR from one mRNA to another and alter the stability characteristics of the recipient mRNA. Certainly, stem-loop structures play important roles in message regulation of histone mRNA [Marzluff and Pandey, 1988], or ferritin and transferrin receptor mRNA regulation [Klausner and Hartford, 1989]. Histone mRNAs are cell cycle regulated and lack a poly (A) tail, but structural information in the 3′ UTR including a 6 base pair stem and 4 base loop motif found in all histone mRNAs, play crucial roles in controlling the rates of translation and degradation. In general, secondary structural features are important because they influence the binding of regulatory proteins that directly or indirectly affect interactions between the mRNA and nucleases and/or because they may act directly as favored recognition sites for particular nuclease activities or as inhibitors of nuclease action.
The genetic changes underlying cancer conversion and progression are accompanied by a decrease in genomic stability of cells [Cifone and Fidler, 1981; Wolman, 1983; Rowley, 1990; Huang et al., 1995a], which leads to heterogeneity of tumor cell populations, alterations in response to chemotherapy, and increased malignant potential. The multitude of changes that are observed during malignant transformation and are most pronounced at advanced stages of the disease, are at least in part due to changes in genome/message stability, as manifested for example by an increased potential for DNA amplification [Rowley, 1990; Wright et al., 1990b; Tlsty, 1990]. Normal diploid cells rarely amplify their DNA, but amplification of oncogenes and genes determining drug resistance is often observed in tumor cell populations, and this is one of the most impressive characteristics that distinguishes normal cells from tumor cells [Wright et al., 1990b; Tlsty, 1990]. The expression of several genes that are known to play fundamental roles in malignant progression are strictly regulated at the posttranscriptional level through mechanisms that control message stability characteristics. Clearly, mechanisms that lead to genomic/message destabilization are important in cancer transformation and progression, and methods are needed for reversing or controlling genomic destabilization which can be utilized in treating cancer.